Project Summary/Abstract The primary monocilium, or cilium, is a specialized organelle that provides a spatially concentrated platform for receipt of extracellular cues and induction of intracellular response for signaling pathways downstream of Sonic Hedgehog (SHH), WNT, PDGFR?, and other extracellular ligands. In non-pathological conditions, single cilia are present on virtually all cell types (except blood cells) in vertebrates. However, ciliary dynamics are frequently altered in cancer, resulting in asymmetry of expression between tumor cells, which typically downregulate cilia, and tumor-associated stroma, which maintain them. This asymmetry supports the ability of cancer cells to condition the growth of surrounding stroma via paracrine SHH signaling, while limiting autocrine SHH responsiveness, and is important in biological processes such as the creation and maintenance of desmoplastic stroma in pancreatic adenocarcinoma (PDAC). Desmoplasia, in turn, promotes tumor resistance to cytotoxic and targeted agents, regulates the immune microenvironment, and otherwise supports aggressive tumor growth. This proposal is based on the idea that better understanding of the mechanisms regulating ciliary integrity and signaling capacity in tumors versus stromal cells, and identification of agents to manipulate these processes, may offer a way to interrupt critical signaling for cancer and improve therapeutic response. We have used candidate and high throughput screening approaches to identify a set of kinase inhibitors of known target specificity (including some FDA agents in clinical use) that caused loss or abnormal retention of cilia. We hypothesize that these drugs, some of which are under investigation for PDAC, may function in part through previously unanticipated activity in enhancing or disrupting tumor-stromal communications. In preliminary studies, we have used siRNA depletion of drug targets for drug hits to confirm and refine understanding of their mechanism of action. We have also shown that some of the drugs regulate cellular response to SHH, revealing entirely novel signaling pathway connections mediated by control of ciliary integrity. The goal of the Aims is to elucidate and target asymmetric cilia-mediated SHH signaling in PDACs. In Aim 1, we will determine how candidate cilia-regulating drugs alter paracellular SHH-initiated communication between pancreatic stellate cells, tumor associated fibroblasts, and PDAC cells. In Aim 2, we will explore in depth the mechanistic role of IRAK4 and other components of the innate immune signaling system we have for the first time identified as mediators of ciliation. This work is expected to provide important insights into novel mechanisms regulating ciliary dynamics, PDAC tumor biology, and the action of currently approved drugs, and may result in entirely new approaches to improve therapeutic effectiveness for PDAC and other cancers.